Eastern Illinois University The Keep Masters Theses Student Theses & Publications 2018 An Efficient Protocol for Root Studies in the Common Sunflower Using Composite Plants Tyler Parks Eastern Illinois University Recommended Citation Parks, Tyler, "An Efficient Protocol for Root Studies in the Common Sunflower Using Composite Plants" (2018). Masters Theses. 4405. https://thekeep.eiu.edu/theses/4405 This Thesis is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. 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GradlMte Candidate Signature Faculty Adviser Signature T�(er furJts )IO-r<i� X:r?/� Printed Name v-27-2t>1rf Date Please submit in duplicate. An Efficient Protocol for Root Studies in the Common Sunflower using Composite Plants (TITLE) BY Tyler Parks THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 2018 YEAR I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE t/-21-lt f/z:t;/1� THESIS COMMITTEECHAIR DATE DATE OR CHAIR'S DESIGNEE THESIS COMMITTEE MEMBER DATE DATE THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER DATE AN EFFICIENT PROTOCOL FOR ROOT STUDIES IN THE COMMON SUNFLOWER USING COMPOSITE PLANTS. Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In The Department of Biological Sciences at Eastern Illinois University By Tyler Parks Charleston Illinois May, 2018 Copyright 2018 by Tyler Parks ii Abstract Sunflower, Helianthus annuus, is one of the most important oilseed crop in the world. Oil from the seeds is prized for its' exceptional quality and flavor. Despite this, sunflowers have been forced onto marginal lands often on semi-arid and non-fertile soil, due to disease pressures and economics making it critical to produce more resilient lines and varieties to withstand these new stresses. The recent availability of the sunflower genome can allow genome-wide characterization of gene families. Stable transformation protocols, which can be used for characterization studies, have been developed for H. annuus using Agrobacterium tumefaciens, but they produce low efficiency and are time­ consuming. For this reason, very few functional studies have been done in the sunflower. Composite plants, produced using Agrobacterium rhizogenes, are plants with transgenic 'hairy' roots and wild type shoots. Composite plants offer benefits over creating fully transgenic plants, namely time and cost. With plant transformation usually being the rate limiting step for gene functionalstudies of non-model plants, composite plants can alleviate this bottleneck. Originally produced in vitro, the method developed here outlines a less expensive exvitro alternative to produce composite sunflowers for study. The critical steps and parameters are outlined and discussed further herein. The testing of more than a dozen genotypes validates the utility and efficiency of this protocol. We also study the performance of three constitutive promoters for the expression of genes of interest. The functional characterization of a root specific transcription factor HaLBD 16, via overexpression and RNAi silencing, further reinforce the utility of the system. iii Acknowledgements With the deepest gratitude, I would like to acknowledge the members of my committee who pushed, pulled, or prodded me along as I attempted to complete my studies. Dr. Yordan Yordanov has been a constant support both in and out of the lab. His high aspirations coupled with realistic, yet challenging expectations are the majority of the reason I accomplished as much as I did. His hands on scientific approach provided me with a myriad of skills and the confidence to achieve any expectations placed on me in the future. He is an outstanding scientist, mentor, and friend. Dr. Barbara Carlsward has been a constant support since my first year at EIU. Her kinds words and sense of humor have been a source of positivity for six years now. Without her, I may have never developed enough of an interest in plants to take on a project such as this one. Dr. Tom Canam has been the person I could rely on to answer questions late into the night in the building. He has always been eager to help, and even stopped what he was working on, to address whatever issue I had. I would also like to thank my lab mates. Without the help and support of my peers I realize that this project would have been much more difficult. I would also like to thank my friends and family for their constant suppo11 despite their lack of knowing fully what it is I did. And lastly, I would like to thank the Department of Biological Sciences for the funding, support, and opportunity they gave me. iv Table of Contents ABSTRACT .......................................................................................... iii Acknowledgen1ents ................................................................................. iv Table of Contents ............................................................................. ........v List of Figures ....................................................................................... viii List of Tables ........................................................................................... x OVERVIEW................... ......... ................................................ .......1 INTRODUCTION...... ......................................................................... .l AIM..................................................................................... ...............3 Chapter 1 Protocol for composite sunflower plants. ........ ... ........... ... ........... 5 1.1 INTRODUCTION... ........................................ ...............................5 1.2 MATERIALS AND METHODS ...... ...................................... .... ........ 8 1.2.l Consumables and Chemicals ................ ...........................................8 1.2.2 Plant Materials .. ............. .............................. ........ ...................... 8 1.2.3 Agrobacterium strain and Plasmid Construct.. ..................................... 8 1.2.4 Seed germination and A. rhizogenes inoculation ............... ..... .............. 9 1.2.S Plant cultivation after hairy root emergence.. .................. ................. 9 1.2.6 P-Glucuronidase detection...... ........... ...... ................. .... ... .......... 10 1.2.7 Statistical Analysis...... .... ............................................ .......